biology Article Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx Shailey Gale Twamley 1,2, Anke Stach 1, Heike Heilmann 3, Berit Söhl-Kielczynski 4, Verena Stangl 1,2, Antje Ludwig 1,2,*,† and Agnieszka Münster-Wandowski 3,*,† 1 Medizinische Klinik für Kardiologie und Angiologie, Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10117 Berlin, Germany; [email protected] (S.G.T.); [email protected] (A.S.); [email protected] (V.S.) 2 DZHK (German Centre for Cardiovascular Research), Partner Site, 10117 Berlin, Germany 3 Institute of Integrative Neuroanatomy, Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10117 Berlin, Germany; [email protected] 4 Institute for Integrative Neurophysiology—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10117 Berlin, Germany; [email protected] * Correspondence: [email protected] (A.L.); [email protected] (A.M.-W.) † These authors equally contributed. Simple Summary: The glycocalyx (GCX) is a hydrated, gel-like layer of biological macromolecules attached to the cell membrane. The GCX acts as a barrier and regulates the entry of external substances into the cell. The function of the GCX is highly dependent on its structure and composition. Citation: Twamley, S.G.; Stach, A.; Pathogenic factors can affect the protective structure of the GCX. We know very little about the three- Heilmann, H.; Söhl-Kielczynski, B.; dimensional organization of the GXC. The tiny and delicate structures of the GCX are difficult Stangl, V.; Ludwig, A.; to study by microscopic techniques. In this study, we evaluated a method to preserve and label Münster-Wandowski, A. Immuno-Electron and Confocal Laser sensitive GCX components with antibodies for high-resolution microscopy analysis. High-resolution Scanning Microscopy of the microscopy is a powerful tool because it allows visualization of ultra-small components and biological Glycocalyx. Biology 2021, 10, 402. interactions. Our method can be used as a tool to better understand the role of the GCX during https://doi.org/10.3390/ the development and progression of diseases, such as viral infections, tumor invasion, and the biology10050402 development of atherosclerosis. Academic Editor: Jukka Finne Abstract: The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and Received: 17 March 2021 diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission Accepted: 1 May 2021 electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by Published: 4 May 2021 conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced Publisher’s Note: MDPI stays neutral antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol with regard to jurisdictional claims in to preserve both antigen accessibility and the unique morphology of the GCX. We established a published maps and institutional affil- iations. combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. components. The presented combination of HPF, FS, rehydration, and immunolabeling for both This article is an open access article TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique distributed under the terms and GCX structure. conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Biology 2021, 10, 402. https://doi.org/10.3390/biology10050402 https://www.mdpi.com/journal/biology Biology 2021, 10, 402 2 of 15 Keywords: glycocalyx; immuno-electron microscopy; high-pressure freezing; freeze-substitution; pre-embedding immunogold labeling; confocal laser scanning microscopy 1. Introduction The glycocalyx (GCX), a carbohydrate rich hydrogel located on the surface of cells, provides mechanical support and forms the selective barrier between the cell surface and the extracellular space [1]. It is involved in many processes that are related to the cell membrane such as blastocyst implantation, embryonic development, leukocyte adhesion, and viral and bacterial infections [2–4]. Glycoproteins (GPs) and proteoglycans (PGs), together with covalently bound glycosaminoglycan (GAG) chains and sialic acid, are the backbone molecules of the GCX. Extending far into the extracellular space, GAG chains such as heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA) are linked and/or intertwined with specific PGs in combination with other ECM proteins [4]. The outer surface layer is more loosely associated with proteins, growth factors, ions, and other biological components [5]. The detailed composition and structure of the GCX varies according to the cell type and determines its functionality under physiological and patho- physiological conditions [4,6]. However, little is known about the structure and dynamics of the GCX in different cell types on an ultrastructural level. This knowledge gap is largely due to a lack of methods that allow both structural preservation and immunolabeling of GCX-specific components. The GCX is a fragile structure, making it difficult to study by TEM and CLSM. Sample preparation by conventional aldehyde fixation followed by alcohol dehydration causes the GCX to collapse, resulting in an incorrect estimation of its dimension and organization [7]. It was established that rapid cryofixation followed by freeze substitution (FS) is a more suitable method for preserving the ultrastructure of the GCX for TEM [8,9]. Ebong and co-workers showed that after rapid freezing (RF) and subsequent FS, the GCX of cultured bovine endothelial cells in the TEM appeared as a several µm thick mesh-like structure, which was clearly different to the collapsed state after chemical fixation [8]. Recently, Poller et al. (2020) used a combined high pressure freezing (HPF) and FS method on human monocytic THP-1 cells, resulting in an equally well-preserved ultrastructure of the GCX [9]. Immuno-electron microscopy is a powerful technique for observing the cellular, sub- cellular, and extracellular localization of antigens and for studying the relationship between antigens and other macromolecules. The rapid cryofixation established for TEM-suitable GCX preservation can be applied before pre- and post-embedding immunolabelling meth- ods [10,11]. In order to stabilize the fragile GCX during resin embedding and polymeriza- tion after rapid cryofixation, the FS media used in the studies of Ebong and Poller contained osmium tetroxide (OsO4)[8,9]. However, since OsO4 cross-links proteins and polypep- tide chains and thus affects antigen–antibody reactions [12], incubation with osmium is generally not recommended prior to immunoreactions. The aim of this study was to provide a method for electron and confocal microscopic analysis of the GCX that warrants both antigen accessibility and morphological preser- vation. We present a combined HPF, FS, rehydration, and pre-embedding immunogold labeling protocol that enables high quality immunocytochemistry while maintaining GCX morphology at the ultrastructural level. 2. Materials and Methods 2.1. Cell Culture To establish our HPF/FS/rehydration and pre-embedding immunolabeling method we used THP-1 cells. THP-1 cells (human acute monocytic leukemia cell line; ATCC, Wesel, ◦ Germany) were cultured in suspension in a humidified incubator at 37 C with 5% CO2 in RPMI-1640 medium (Invitrogen, Karlsruhe, Germany), supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany), 100 U/mL penicillin, 100 µg/mL streptomycin Biology 2021, 10, 402 3 of 15 (Invitrogen), and 2 mM L-glutamine (Invitrogen). Exact cell numbers were determined with a hemocytometer. 2.2. Formalin Fixation THP-1 cells were washed with phosphate buffered saline (PBS, Thermo Fisher, Waltham, MA, USA) and fixed in phosphate buffered 4% formalin solution (Roti-Histofix, Carl Roth GmbH, Karlsruhe, Germany) for 10 min at room temperature (RT). Samples were washed with PBS and blocked for 1 h at room temperature in PBS with 5% normal goat serum (NGS) and 2% bovine serum albumin (BSA) (blocking buffer). Primary antibodies (Table1 , CD44 (IM7) FITC, CS-56) were incubated overnight at 4 ◦C in the dark in blocking buffer. Next, samples that were incubated with primary antibodies for CS were washed and incubated with secondary antibody (goat anti-mouse IgM Alexa Fluor-488; Invitrogen, Karlsruhe, Germany) suspended in PBS with 2% NGS overnight at 4 ◦C. After immunofluorescence labeling was complete, cells were stained for 5 min in the